AGR2调控IFITM3的表达促进肝癌细胞的增殖
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摘要
目的:探讨重组人前梯度同源蛋白2(anterior gradient homolog 2,AGR2)的表达对肝癌细胞增殖能力的影响及其可能的作用机制.方法:通过实时荧光定量PCR、Western blot、免疫组织化学检测40例肝癌患者癌组织和对应癌旁组织中AGR2基因、干扰素诱导跨膜蛋白3(interferon-induced transmembrane protein 3,IFITM3)的表达情况,构建AGR2的过表达质粒pcDNA3.1-AGR2,并将其转染至肝癌细胞中,运用实时荧光定量PCR和蛋白质印迹法分别检测AGR2 mRNA和蛋白的表达,CCK-8(cell counting kit 8)法检测细胞的增殖能力,利用流式细胞技术检测细胞凋亡,蛋白质印迹法检测IFITM3的表达.结果:在原发性肝癌组织中AGR2,IFITM3呈现高表达;成功构建AGR2的过表达质粒pcDNA3.1-AGR2,成功构建稳定高表达AGR2的肝癌HepG2细胞.pcDNA3.1-AGR2转染组HepG2细胞中AGR2 mRNA和蛋白的表达水平明显高于阴性对照组和空白对照组.与阴性对照组比较,pcDNA3.1-AGR2转染组HepG2细胞的体外增殖能力和克隆形成能力明显升高,IFITM3蛋白的表达水平也随之升高.结论:上调AGR2可以增加IFITM3的表达,促进肝癌细胞的增殖.
AIM:To investigate the effect of anterior gradient homolog 2(AGR2) expression on the proliferation of hepatocellular carcinoma cells,and to explore the possible mechanism.METHODS:The expression of AGR2 and interferon-induced transmembrane protein3(IFITM3) in hepatocellular carcinoma and adjacent tissues was detected by fluorescence quantitative PCR and Western blot.The expression of AGR2 mRNA and protein was examined by real-time fluorescence quantitative PCR and Western blot after transfection with pcDNA3.1-AGR2.The cellular growth ability was examined by CCK-8 assay,and the colony formation ability was detected by colony formation assay.Flow cytometry assay was used to determine the apoptosis index.The expression level of IFITM3 protein was examined by Western blot.RESULTS:AGR2 and IFITM3 mRNA and protein expression in hepatocellular carcinoma tissues was significantly higher than that in the corresponding adjacent tissues.The pcDNA3.1-AGR2 was successfully constructed,and HepG2 cells with stable expression of AGR2 were established.The expression levels of AGR2 mRNA and protein in HepG2 cells after transfection with pcDNA3.1-AGR2 were higher than those in the negative control cells,and the cellular growth ability of HepG2 cells after transfection with pcDNA3.1-AGR2 was significantly increased.IFITM3 protein expression was also increased after transfection with pcDNA3.1-AGR2.CONCLUSION:Raised AGR2 expression can increase the expression of IFITM3 and promote the proliferation of hepatocellular cells.
AIM:To investigate the effect of anterior gradient homolog 2(AGR2) expression on the proliferation of hepatocellular carcinoma cells,and to explore the possible mechanism.METHODS:The expression of AGR2 and interferon-induced transmembrane protein3(IFITM3) in hepatocellular carcinoma and adjacent tissues was detected by fluorescence quantitative PCR and Western blot.The expression of AGR2 mRNA and protein was examined by real-time fluorescence quantitative PCR and Western blot after transfection with pcDNA3.1-AGR2.The cellular growth ability was examined by CCK-8 assay,and the colony formation ability was detected by colony formation assay.Flow cytometry assay was used to determine the apoptosis index.The expression level of IFITM3 protein was examined by Western blot.RESULTS:AGR2 and IFITM3 mRNA and protein expression in hepatocellular carcinoma tissues was significantly higher than that in the corresponding adjacent tissues.The pcDNA3.1-AGR2 was successfully constructed,and HepG2 cells with stable expression of AGR2 were established.The expression levels of AGR2 mRNA and protein in HepG2 cells after transfection with pcDNA3.1-AGR2 were higher than those in the negative control cells,and the cellular growth ability of HepG2 cells after transfection with pcDNA3.1-AGR2 was significantly increased.IFITM3 protein expression was also increased after transfection with pcDNA3.1-AGR2.CONCLUSION:Raised AGR2 expression can increase the expression of IFITM3 and promote the proliferation of hepatocellular cells.
引文
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2 Gory I,Fink M,Bell S,Gow P,Nicoll A,Knight V,Dev A,Rode A,Bailey M,Cheung W,Kemp W,Roberts SK.Radiofrequency ablation versus resection for the treatment of early stage hepatocellular carcinoma:a multicenter Australian study.Scand J Gastroenterol 2015;50:567-576[PMID:25615260 DOI:10.3109/00365521.2014.953572]
3李涵,王进,杨丽敏,宁寒冰.人前梯度蛋白2在结肠癌组织中的表达及其意义.世界华人消化杂志2014;22:1064-1069
4 Bergstrom JH,Berg KA,Rodriguez-Pineiro AM,Stecher B,Johansson ME,Hansson GC.AGR2,an endoplasmic reticulum protein,is secreted into the gastrointestinal mucus.PLoS One 2014;9:e104186[PMID:25111734 DOI:10.1371/journal.pone.0104186]
5 Tsuji T,Satoyoshi R,Aiba N,Kubo T,Yanagihara K,Maeda D,Goto A,Ishikawa K,Yashiro M,Tanaka M.Agr2 mediates paracrine effects on stromal fibroblasts that promote invasion by gastric signet-ring carcinoma cells.Cancer Res 2015;75:356-366[PMID:25488752 DOI:0.1158/0008-5472.CAN-14-1693]
6 Mizuuchi Y,Aishima S,Ohuchida K,Shindo K,Fujino M,Hattori M,Miyazaki T,Mizumoto K,Tanaka M,Oda Y.Anterior gradient 2downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial-mesenchymal transition.Lab Invest2015;95:193-206[PMID:25418581 DOI:10.1038/labinvest.2014.138]
7 Chutiwitoonchai N,Hiyoshi M,HiyoshiYoshidomi Y,Hashimoto M,Tokunaga K,Suzu S.Characteristics of IFITM,the newly identified IFN-inducible anti-HIV-1 family proteins.Microbes Infect 2013;15:280-290[PMID:23376165 DOI:10.1016/j.micinf.2012.12.003]
8 Diamond MS,Farzan M.The broad-spectrum antiviral functions of IFIT and IFITM proteins.Nat Rev Immunol 2013;13:46-57[PMID:23237964 DOI:10.1038/nri3344]
9 Shen C,Wu XR,Jiao WW,Sun L,Feng WX,Xiao J,Miao Q,Liu F,Yin QQ,Zhang CG,Guo YJ,Shen AD.A functional promoter polymorphism of IFITM3 is associated with susceptibility to pediatric tuberculosis in Han Chinese population.PLoS One 2013;8:e67816[PMID:23874452 DOI:10.1371/journal.pone.0067816]
10 Bailey CC,Zhong G,Huang IC,Farzan M.IFITM-Family Proteins:The Cell's First Line of Antiviral Defense.Annu Rev Virol 2014;1:261-283[PMID:25599080 DOI:10.1146/annurevvirology-031413-085537]
11 Wakim LM,Gupta N,Mintern JD,Villadangos JA.Enhanced survival of lung tissue-resident memory CD8+T cells during infection with influenza virus due to selective expression of IFITM3.Nat Immunol2013;14:238-245[PMID:23354485 DOI:10.1038/ni.2525]
12 Wright TM,Wardell SE,Jasper JS,Stice JP,Safi R,Nelson ER,McDonnell DP.Delineation of a FOXA1/ERa/AGR2 regulatory loop that is dysregulated in endocrine therapy-resistant breast cancer.Mol Cancer Res 2014;12:1829-1839[PMID:25100862 DOI:10.1158/1541-7786.MCR-14-0195]
13 Liu D,Rudland PS,Sibson DR,Platt-Higgins A,Barraclough R.Human homologue of cement gland protein,a novel metastasis inducer associated with breast carcinomas.Cancer Res2005;65:3796-3805[PMID:15867376 DOI:10.1158/0008-5472.CAN-04-3823]
14 Li Y,Lu J,Peng Z,Tan G,Liu N,Huang D,Zhang Z,Duan C,Tang X,Tang F.N,N'-dinitrosopiperazinemediated AGR2 is involved in metastasis of nasopharyngeal carcinoma.PLoS One 2014;9:e92081[PMID:24717913 DOI:10.1371/journal.pone.0092081]
15 Niederreiter L,Kaser A.Endoplasmic reticulum stress and inflammatory bowel disease.Acta Gastroenterol Belg 2011;74:330-333[PMID:21861319]
16 Di Maro G,Salerno P,Unger K,Orlandella FM,Monaco M,Chiappetta G,Thomas G,OczkoWojciechowska M,Masullo M,Jarzab B,Santoro M,Salvatore G.Anterior gradient protein 2 promotes survival,migration and invasion of papillary thyroid carcinoma cells.Mol Cancer 2014;13:160[PMID:24976026 DOI:10.1186/1476-4598-13-160]
17 Pizzi M,Fassan M,Realdon S,Balistreri M,Battaglia G,Giacometti C,Zaninotto G,Zagonel V,De Boni M,Rugge M.Anterior gradient 2 profiling in Barrett columnar epithelia and adenocarcinoma.Hum Pathol 2012;43:1839-1844[PMID:22521076DOI:10.1016/j.humpath.2012.01.004]
18 Huang J,Wang L,Jiang M,Chen Q,Jiang Z,Feng H.AGR2-mediated lung adenocarcinoma metastasis novel mechanism network through repression with interferon coupling cytoskeleton to steroid metabolism-dependent humoral immune response.Cell Immunol 2014;290:102-106[PMID:24960290 DOI:10.1016/j.cellimm.2014.05.008]
19 Salmans ML,Zhao F,Andersen B.The estrogenregulated anterior gradient 2(AGR2)protein in breast cancer:a potential drug target and biomarker.Breast Cancer Res 2013;15:204[PMID:23635006 DOI:10.1186/bcr3408]
20 Sung HY,Choi EN,Lyu D,Park AK,Ju W,Ahn JH.Aberrant hypomethylation-mediated AGR2overexpression induces an aggressive phenotype in ovarian cancer cells.Oncol Rep 2014;32:815-820[PMID:24920423 DOI:10.3892/or.2014.3243]
21 Riener MO,Thiesler T,Hellerbrand C,Amann T,Cathomas G,Fritzsche FR,Dahl E,Bahra M,Weichert W,Terracciano L,Kristiansen G.Loss of anterior gradient-2 expression is an independent prognostic factor in colorectal carcinomas.Eur J Cancer 2014;50:1722-1730[PMID:24794000 DOI:10.1016/j.ejca.2014.04.012]
22 Andreu P,Colnot S,Godard C,Laurent-Puig P,Lamarque D,Kahn A,Perret C,Romagnolo B.Identification of the IFITM family as a new molecular marker in human colorectal tumors.Cancer Res 2006;66:1949-1955[PMID:16488993DOI:10.1158/0008-5472.CAN-05-2731]
23 Li D,Peng Z,Tang H,Wei P,Kong X,Yan D,Huang F,Li Q,Le X,Li Q,Xie K.KLF4-mediated negative regulation of IFITM3 expression plays a critical role in colon cancer pathogenesis.Clin Cancer Res 2011;17:3558-3568[PMID:21531817DOI:10.1158/1078-0432.CCR-10-2729]
24 Xue WW,Wang HN,Wang ZM,Qiu MX,Che J,Deng FJ,Liu JD.Cloning and characterization of ifitm1 and ifitm3 expression during early zebrafish development.Zygote 2015 Jan 23.[Epub ahead of print][PMID:25613417 DOI:10.1017/S0967199414000756]
25 Chesarino NM,McMichael TM,Yount JS.Regulation of the trafficking and antiviral activity of IFITM3 by post-translational modifications.Future Microbiol 2014;9:1151-1163[PMID:25405885 DOI:10.2217/fmb.14.65]
26 Williams DE,Wu WL,Grotefend CR,Radic V,Chung C,Chung YH,Farzan M,Huang IC.IFITM3polymorphism rs12252-C restricts influenza A viruses.PLoS One 2014;9:e110096[PMID:25314048 DOI:10.1371/journal.pone.0110096]
27 Hu J,Wang S,Zhao Y,Guo Q,Zhang D,Chen J,Li J,Fei Q,Sun Y.Mechanism and biological significance of the overexpression of IFITM3 in gastric cancer.Oncol Rep 2014;32:2648-2656[PMID:25270246 DOI:10.3892/or.2014.3522]
28 Yu M,Qi W,Huang Z,Zhang K,Ye J,Liu R,Wang H,Ma Y,Liao M,Ning Z.Expression profile and histological distribution of IFITM1 and IFITM3during H9N2 avian influenza virus infection in BALB/c mice.Med Microbiol Immunol 2014 Sep 30.[Epub ahead of print][PMID:25265877 DOI:10.1007/s00430-014-0361-2]
29 Zhu X,He Z,Yuan J,Wen W,Huang X,Hu Y,Lin C,Pan J,Li R,Deng H,Liao S,Zhou R,Wu J,Li J,Li M.IFITM3-containing exosome as a novel mediator for anti-viral response in dengue virus infection.Cell Microbiol 2015;17:105-118[PMID:25131332DOI:10.1111/cmi.12339]
30 Fan J,Peng Z,Zhou C,Qiu G,Tang H,Sun Y,Wang X,Li Q,Le X,Xie K.Gene-expression profiling in Chinese patients with colon cancer by coupling experimental and bioinformatic genomewide geneexpression analyses:identification and validation of IFITM3 as a biomarker of early colon carcinogenesis.Cancer 2008;113:266-275[PMID:18470904 DOI:10.1002/cncr.23551]
31 Yang M,Gao H,Chen P,Jia J,Wu S.Knockdown of interferon-induced transmembrane protein 3expression suppresses breast cancer cell growth and colony formation and affects the cell cycle.Oncol Rep 2013;30:171-178[PMID:23624618 DOI:10.3892/or.2013.2428]
2 Gory I,Fink M,Bell S,Gow P,Nicoll A,Knight V,Dev A,Rode A,Bailey M,Cheung W,Kemp W,Roberts SK.Radiofrequency ablation versus resection for the treatment of early stage hepatocellular carcinoma:a multicenter Australian study.Scand J Gastroenterol 2015;50:567-576[PMID:25615260 DOI:10.3109/00365521.2014.953572]
3李涵,王进,杨丽敏,宁寒冰.人前梯度蛋白2在结肠癌组织中的表达及其意义.世界华人消化杂志2014;22:1064-1069
4 Bergstrom JH,Berg KA,Rodriguez-Pineiro AM,Stecher B,Johansson ME,Hansson GC.AGR2,an endoplasmic reticulum protein,is secreted into the gastrointestinal mucus.PLoS One 2014;9:e104186[PMID:25111734 DOI:10.1371/journal.pone.0104186]
5 Tsuji T,Satoyoshi R,Aiba N,Kubo T,Yanagihara K,Maeda D,Goto A,Ishikawa K,Yashiro M,Tanaka M.Agr2 mediates paracrine effects on stromal fibroblasts that promote invasion by gastric signet-ring carcinoma cells.Cancer Res 2015;75:356-366[PMID:25488752 DOI:0.1158/0008-5472.CAN-14-1693]
6 Mizuuchi Y,Aishima S,Ohuchida K,Shindo K,Fujino M,Hattori M,Miyazaki T,Mizumoto K,Tanaka M,Oda Y.Anterior gradient 2downregulation in a subset of pancreatic ductal adenocarcinoma is a prognostic factor indicative of epithelial-mesenchymal transition.Lab Invest2015;95:193-206[PMID:25418581 DOI:10.1038/labinvest.2014.138]
7 Chutiwitoonchai N,Hiyoshi M,HiyoshiYoshidomi Y,Hashimoto M,Tokunaga K,Suzu S.Characteristics of IFITM,the newly identified IFN-inducible anti-HIV-1 family proteins.Microbes Infect 2013;15:280-290[PMID:23376165 DOI:10.1016/j.micinf.2012.12.003]
8 Diamond MS,Farzan M.The broad-spectrum antiviral functions of IFIT and IFITM proteins.Nat Rev Immunol 2013;13:46-57[PMID:23237964 DOI:10.1038/nri3344]
9 Shen C,Wu XR,Jiao WW,Sun L,Feng WX,Xiao J,Miao Q,Liu F,Yin QQ,Zhang CG,Guo YJ,Shen AD.A functional promoter polymorphism of IFITM3 is associated with susceptibility to pediatric tuberculosis in Han Chinese population.PLoS One 2013;8:e67816[PMID:23874452 DOI:10.1371/journal.pone.0067816]
10 Bailey CC,Zhong G,Huang IC,Farzan M.IFITM-Family Proteins:The Cell's First Line of Antiviral Defense.Annu Rev Virol 2014;1:261-283[PMID:25599080 DOI:10.1146/annurevvirology-031413-085537]
11 Wakim LM,Gupta N,Mintern JD,Villadangos JA.Enhanced survival of lung tissue-resident memory CD8+T cells during infection with influenza virus due to selective expression of IFITM3.Nat Immunol2013;14:238-245[PMID:23354485 DOI:10.1038/ni.2525]
12 Wright TM,Wardell SE,Jasper JS,Stice JP,Safi R,Nelson ER,McDonnell DP.Delineation of a FOXA1/ERa/AGR2 regulatory loop that is dysregulated in endocrine therapy-resistant breast cancer.Mol Cancer Res 2014;12:1829-1839[PMID:25100862 DOI:10.1158/1541-7786.MCR-14-0195]
13 Liu D,Rudland PS,Sibson DR,Platt-Higgins A,Barraclough R.Human homologue of cement gland protein,a novel metastasis inducer associated with breast carcinomas.Cancer Res2005;65:3796-3805[PMID:15867376 DOI:10.1158/0008-5472.CAN-04-3823]
14 Li Y,Lu J,Peng Z,Tan G,Liu N,Huang D,Zhang Z,Duan C,Tang X,Tang F.N,N'-dinitrosopiperazinemediated AGR2 is involved in metastasis of nasopharyngeal carcinoma.PLoS One 2014;9:e92081[PMID:24717913 DOI:10.1371/journal.pone.0092081]
15 Niederreiter L,Kaser A.Endoplasmic reticulum stress and inflammatory bowel disease.Acta Gastroenterol Belg 2011;74:330-333[PMID:21861319]
16 Di Maro G,Salerno P,Unger K,Orlandella FM,Monaco M,Chiappetta G,Thomas G,OczkoWojciechowska M,Masullo M,Jarzab B,Santoro M,Salvatore G.Anterior gradient protein 2 promotes survival,migration and invasion of papillary thyroid carcinoma cells.Mol Cancer 2014;13:160[PMID:24976026 DOI:10.1186/1476-4598-13-160]
17 Pizzi M,Fassan M,Realdon S,Balistreri M,Battaglia G,Giacometti C,Zaninotto G,Zagonel V,De Boni M,Rugge M.Anterior gradient 2 profiling in Barrett columnar epithelia and adenocarcinoma.Hum Pathol 2012;43:1839-1844[PMID:22521076DOI:10.1016/j.humpath.2012.01.004]
18 Huang J,Wang L,Jiang M,Chen Q,Jiang Z,Feng H.AGR2-mediated lung adenocarcinoma metastasis novel mechanism network through repression with interferon coupling cytoskeleton to steroid metabolism-dependent humoral immune response.Cell Immunol 2014;290:102-106[PMID:24960290 DOI:10.1016/j.cellimm.2014.05.008]
19 Salmans ML,Zhao F,Andersen B.The estrogenregulated anterior gradient 2(AGR2)protein in breast cancer:a potential drug target and biomarker.Breast Cancer Res 2013;15:204[PMID:23635006 DOI:10.1186/bcr3408]
20 Sung HY,Choi EN,Lyu D,Park AK,Ju W,Ahn JH.Aberrant hypomethylation-mediated AGR2overexpression induces an aggressive phenotype in ovarian cancer cells.Oncol Rep 2014;32:815-820[PMID:24920423 DOI:10.3892/or.2014.3243]
21 Riener MO,Thiesler T,Hellerbrand C,Amann T,Cathomas G,Fritzsche FR,Dahl E,Bahra M,Weichert W,Terracciano L,Kristiansen G.Loss of anterior gradient-2 expression is an independent prognostic factor in colorectal carcinomas.Eur J Cancer 2014;50:1722-1730[PMID:24794000 DOI:10.1016/j.ejca.2014.04.012]
22 Andreu P,Colnot S,Godard C,Laurent-Puig P,Lamarque D,Kahn A,Perret C,Romagnolo B.Identification of the IFITM family as a new molecular marker in human colorectal tumors.Cancer Res 2006;66:1949-1955[PMID:16488993DOI:10.1158/0008-5472.CAN-05-2731]
23 Li D,Peng Z,Tang H,Wei P,Kong X,Yan D,Huang F,Li Q,Le X,Li Q,Xie K.KLF4-mediated negative regulation of IFITM3 expression plays a critical role in colon cancer pathogenesis.Clin Cancer Res 2011;17:3558-3568[PMID:21531817DOI:10.1158/1078-0432.CCR-10-2729]
24 Xue WW,Wang HN,Wang ZM,Qiu MX,Che J,Deng FJ,Liu JD.Cloning and characterization of ifitm1 and ifitm3 expression during early zebrafish development.Zygote 2015 Jan 23.[Epub ahead of print][PMID:25613417 DOI:10.1017/S0967199414000756]
25 Chesarino NM,McMichael TM,Yount JS.Regulation of the trafficking and antiviral activity of IFITM3 by post-translational modifications.Future Microbiol 2014;9:1151-1163[PMID:25405885 DOI:10.2217/fmb.14.65]
26 Williams DE,Wu WL,Grotefend CR,Radic V,Chung C,Chung YH,Farzan M,Huang IC.IFITM3polymorphism rs12252-C restricts influenza A viruses.PLoS One 2014;9:e110096[PMID:25314048 DOI:10.1371/journal.pone.0110096]
27 Hu J,Wang S,Zhao Y,Guo Q,Zhang D,Chen J,Li J,Fei Q,Sun Y.Mechanism and biological significance of the overexpression of IFITM3 in gastric cancer.Oncol Rep 2014;32:2648-2656[PMID:25270246 DOI:10.3892/or.2014.3522]
28 Yu M,Qi W,Huang Z,Zhang K,Ye J,Liu R,Wang H,Ma Y,Liao M,Ning Z.Expression profile and histological distribution of IFITM1 and IFITM3during H9N2 avian influenza virus infection in BALB/c mice.Med Microbiol Immunol 2014 Sep 30.[Epub ahead of print][PMID:25265877 DOI:10.1007/s00430-014-0361-2]
29 Zhu X,He Z,Yuan J,Wen W,Huang X,Hu Y,Lin C,Pan J,Li R,Deng H,Liao S,Zhou R,Wu J,Li J,Li M.IFITM3-containing exosome as a novel mediator for anti-viral response in dengue virus infection.Cell Microbiol 2015;17:105-118[PMID:25131332DOI:10.1111/cmi.12339]
30 Fan J,Peng Z,Zhou C,Qiu G,Tang H,Sun Y,Wang X,Li Q,Le X,Xie K.Gene-expression profiling in Chinese patients with colon cancer by coupling experimental and bioinformatic genomewide geneexpression analyses:identification and validation of IFITM3 as a biomarker of early colon carcinogenesis.Cancer 2008;113:266-275[PMID:18470904 DOI:10.1002/cncr.23551]
31 Yang M,Gao H,Chen P,Jia J,Wu S.Knockdown of interferon-induced transmembrane protein 3expression suppresses breast cancer cell growth and colony formation and affects the cell cycle.Oncol Rep 2013;30:171-178[PMID:23624618 DOI:10.3892/or.2013.2428]